Design, synthesis and in vitro and in vivo antitumour activity of 3-benzylideneindolin-2-one derivatives, a novel class of small-molecule inhibitors of the MDM2-p53 interaction

Article abstract of DOI:10.1016/j.ejmech.2014.05.027

A novel class of small-molecule inhibitors of MDM2-p53 interaction with a (E)-3-benzylideneindolin-2-one scaffold was identified using an integrated virtual screening strategy that combined both pharmacophore- and structure-based approaches. The hit optimisation identified several compounds with more potent activity than the hit compound and the positive drug nutlin-3a, especially compound 1b, which exhibited both the highest binding affinity to MDM2 (K _i = 0.093 μM) and the most potent antiproliferative activity against HCT116 (wild type p53) cells (GI₅₀ = 13.42 μM). Additionally, 1b dose-dependently inhibited tumour growth in BALB/c mice bearing CT26 colon carcinoma, with no visible sign of toxicity. In summary, compound 1b represents a novel and promising lead structure for the development of anticancer drugs as MDM2-p53 interaction disruptors.

Full text of DOI:10.1016/j.ejmech.2014.05.027

Accepted Manuscript
Design, synthesis and in vitro and in vivo antitumor activity of 3-benzylideneindolin-2-
one derivatives, a novel class of small-molecule inhibitors of the MDM2-p53
interaction
Guang-hui Zheng, Jia-jia Shen, Yue-chen Zhan, Hong Yi, Si-tu Xue, Zhen Wang,
Xing-yue Ji, Zhuo-rong Li
PII:
S0223-5234(14)00441-3
10.1016/j.ejmech.2014.05.027
EJMECH 6983
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 November 2013
Revised Date: 5 May 2014
Accepted Date: 6 May 2014
Please cite this article as: G.-h. Zheng, J.-j. Shen, Y.-c. Zhan, H. Yi, S.-t. Xue, Z. Wang, X.-y. Ji, Z.-r. Li,
Design, synthesis and in vitro and in vivo antitumor activity of 3-benzylideneindolin-2-one derivatives,
a novel class of small-molecule inhibitors of the MDM2-p53 interaction, European Journal of Medicinal
Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.027.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and in vitro and in vivo antitumor activity of
3
-benzylideneindolin-2-one derivatives, a novel class of small-molecule inhibitors
of the MDM2-p53 interaction
Guang-hui Zheng, Jia-jia Shen, Yue-chen Zhan, Hong Yi, Si-tu Xue, Zhen Wang,
**
*
Xing-yue Ji , and Zhuo-rong Li
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and
Peking Union Medical College, Beijing 100050, People’s Republic of China
*
Corresponding author: Tel.: +86-10-63027185; Fax: +86-10-63017302; E-mail:
l-z-r@263.net.
**
Corresponding author. Tel.: +86-10-63151107; Fax: +86-10-63017302; E-mail:
jixingyue@imb.pumc.edu.cn.
Abstract: A novel class of small-molecule inhibitors of MDM2-p53 interaction with a
(E)-3-benzylideneindolin-2-one scaffold was identified using an integrated virtual
screening strategy that combined both pharmacophore- and structure-based
approaches. The hit optimisation identified several compounds with more potent
activity than the hit compound and the positive drug nutlin-3a, especially compound
1
b, which exhibited both the highest binding affinity to MDM2 (K = 0.093 µM) and
i
the most potent antiproliferative activity against HCT116 (wild type p53) cells (GI =
5
0
1
3.42 µM). Additionally, 1b dose-dependently inhibited tumor growth in BALB/c
mice bearing CT26 colon carcinoma, with no visible sign of toxicity. In summary,
compound 1b represents a novel and promising lead structure for the development of
anticancer drugs as MDM2-p53 interaction disruptors.
Keywords: small-molecule inhibitors; MDM2-p53 interaction; antiproliferative
activity;
1
. Introduction
The tumor suppressor protein p53 plays a pivotal role in DNA repair, cell cycle
regulation and apoptosis [1, 2]. Moreover, this protein is an attractive anticancer
therapeutic target because it can be functionally activated to eradicate tumor. It has
1

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been established that the p53 protein is mutated or deleted in half of human cancers.
In the remaining cases, p53 retains its wild type form, but its activity is inhibited by
the human murine double minute 2 (MDM2, also known as HDM2) oncoprotein via
the following mechanisms: a) binding to the p53 transactivation domain to inhibit its
transcriptional activity, b) exporting p53 out of the nucleus, and c) promoting
proteasome-mediated degradation of p53 [3]. Consequently, blocking the MDM2-p53
interaction to reactivate p53 is a promising anticancer therapeutic strategy.
One successful approach for targeting protein-protein interactions (PPIs) is the
rational design of small-molecules that mimic the interaction of a few key residues
(i.e., hot spots) at the protein-protein interface [4]. Unlike common PPIs, in which the
interface is large, shallow and nondescript [5], the MDM2-p53 interface consists of a
cluster of narrow and deep hydrophobic clefts that could be targeted by
small-molecule inhibitors. Mutational analysis and alanine scanning of p53 peptides
revealed that the Phe19, Trp23 and Leu26 residues (hot spots) play an important role
in the interaction between p53 and MDM2 (Figure 1) [6]. Hence, these residues could
be used as a template to design small-molecule inhibitors. To date, there are over 20
different chemotypes that have been shown to antagonise the MDM2-p53 interaction,
including nutlins [7], benzodiazepinediones [8], isoindolinones [9], chalcones [10],
piperidinones [11], pyrrolidones [12], pyrrolopyrimidines [13], spiro-oxindoles [14],
and chromenotriazolopyrimidine [15], xanthones [16]. Among these, the nutlins,
spiro-oxindoles (MI derivatives), and piperidinones (AM-8553) have shown the most
potential for development as clinical agents to treat cancer (Figure 1). Although these
compounds share different scaffold structures, they all inhibit the MDM2-p53
interaction by mimicking the three hot spots residues of p53 (Figure 1).
The nutlins were identified as the first class of potent, non-peptide, specific
inhibitors of the MDM2-p53 interaction by high throughput screening [7]. One of
nutlin derivatives, nutlin-3a, binds with high affinity to MDM2, and it is orally active
with no visible sign of toxicity in mouse models of human cancer containing
wild-type p53 [7, 17]. One nutlin derivative, RO5045337, has already entered phase I
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clinical trials [18]. Using a computational structure-based de novo design strategy,
Wang’s group discovered a new class of inhibitors of the MDM2-p53 interaction,
spiro-oxindoles [14]. Further optimisation lead to the compound MI-219, which has
potent inhibitory activity (K = 13.2 nM) and superior pharmacokinetics (PK) profiles
i
(oral bioavailability F = 65%) [19]. Compound MI-773, which was derived from this
scaffold, has also entered phase I clinical trials [18]. Recently, another promising
inhibitor, AM-8553, containing a piperidinone scaffold was designed using a
structure-based de novo strategy. This compound was reported to have potent activity
both in vitro and vivo, along with an oral bioavailability of 100% in rats [11].
In summary, the interaction between MDM2 and p53 represents a promising
target for the development of novel anticancer drugs that could be targeted by
small-molecule inhibitors. To date, although over 20 small-molecule inhibitors with a
diversity array scaffolds have been discovered, , around 6 candidate compounds with
potent inhibitory activity and superior PK profiles have entered clinical development
[
18]. It would therefore be highly desirable to discover a novel small-molecule
inhibitor with potent activity and an improved ADMET profile.
In this study, we sought to discover novel small-molecule inhibitors of the
MDM2-p53 interaction with potent affinity. We identified a novel class of
MDM2-p53 inhibitors with a 3-benzylidene-indolin-2-one scaffold by combining both
pharmacophore- and structure-based methods as a virtual screening strategy. Hit
optimisation was also conducted, and the preliminary structure-activity relationships
were assessed. Additionally, a compound with relatively potent activity in vitro was
chosen to further assay its antitumor activity in vivo.
2
2
. Results and discussion
.1 Virtual screening and hit identification
Computer-aided drug design (CADD) plays an indispensable role both in the
discovery and optimisation of hit/lead compounds. Currently, two computational 3D
database-screening approaches are available, pharmacophore- and structure-based
approaches. The primary advantages of the pharmacophore-based method are its high
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screening speed and rapid elimination of compounds without critical binding elements.
However, this method suffers from a high false positive rate because it does not
consider the receptor. Alternatively, the structure-based method (docking) can
quantitatively assess the binding affinity of a compound for its target protein, but this
process is rather time-consuming. Clearly, these two methods complement each other
in terms of screening speed and accuracy of screening results; hence, combining these
two complementary approaches is a valid screening strategy. [20, 21]
In this study, we employed an integrated screening strategy combining
pharmacophore- and structure-based methods. It is widely accepted that the
interaction between inhibitors and MDM2 primarily involve three hydrophobic
pockets (Phe19, Trp23 and Leu26). Based on this information, a simple
pharmacophore model was constructed manually in Discovery studio 3.0 (DS 3.0).
First, the crystal structure of a known inhibitor (Pip-1, a derivative of the
piperidinones) complexed with MDM2 (PDB ID: 2LZG) was employed as reference,
and four hydrophobic features (Figure 2, blue spheres) were constructed manually
based on its interaction with MDM2. Additionally, to avoid receptor interactions, the
α-C atoms of the amino acids within 5 Å of the ligand were selected and marked as
exclusion volumes (Figure 2, grey spheres).
To validate the generated pharmacophore hypotheses, a test set consisting of 16
active compounds and 48 inactive compounds was collected. The active compounds
were all extracted from the PDB database and the reported literature, and the inactive
compounds, also known as the decoy set, were derived according to Wallach’s
method [22]. The compounds in the decoy set had to satisfy the following constraints
with respect to the reference drug (Pip-1): a) molecular weight of ±40 Da; b) the exact
same number of rotational bonds, hydrogen bond donors (HBDs), and hydrogen bond
acceptors (HBAs); c) a CLogP of ±1.0 (ALogP was used here); and d) a Tanimoto
coefficient of less than 0.9. The “Screen library” and “Calculate ROC Curve”
modules in DS 3.0 were used, and 15 of 16 inhibitors were enriched from the test set;
however, 12 non-inhibitors were also retrieved (false positives). The area under the
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ROC curve was 0.798, and the ROC evaluation was “fair”. These results indicate that
this pharmacophore was sufficiently good to be used as a 3D query to retrieve
potential hit compounds.
The validated pharmacophore hypothesis was then used as a 3D query for
retrieving potential inhibitors from S406-SETA, S406-SETB, Maybridge and the NCI
database in Catalyst. The “Screen library” module in DS 3.0 was used. The
“Conformation generation” was set to “fast”, and the other parameters were set as
default. The identified compounds were further filtered by the Lipinski’s rule of five.
Lastly, a total of approximately 6000 compounds were retrieved for further docking
studies (structure-based approach).
2
.2 Docking study and biological assay in vitro
To further verify the compounds identified by pharmacophore mapping, the 6000
retrieved compounds were docked into the p53 binding pockets of MDM2. The
protein structure PDB ID 3LBL was chosen as the reference receptor because its
ligand had high binding affinity (K = 32 nM) and high resolution (1.6 Å). Docking
i
was performed using sybyl 7.3, and the Surflex-Dock module (SFXC) was employed.
All the parameters were set as default. After docking, the potential hits were
cherry-picked for synthesis and biological assays in vitro based on their docking
scores, scaffold diversity, synthesizability, and most importantly, their docking poses.
Based on their docking poses predicted by SFXC, we performed a visual inspection to
confirm that all the chosen potential hits mimicked the three hot spot residues of p53
and occupied the three main hydrophobic pockets of MDM2. Ultimately, 10
compounds with diversity of scaffolds (Please refer to supplementary data) were
selected for synthesis, and each was subjected to fluorescence polarization assay to
validate their inhibitory activity against the MDM2-p53 interaction; nutlin-3a, which
is one of the most potent inhibitors of the MDM2-p53 interaction, was used as the
positive control.
Fortunately, one compound (4b, Figure 3) showed relatively high binding affinity
5

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(
K = 0.23 µM) to MDM2 with (E)-3-benzylideneindolin-2-one scaffold, and hence
i
compound 4b was identified as hit compound for further optimisation.
2
.3 Design
To gain a better understanding of the interaction between compound 4b and
MDM2, we proposed a possible binding mode of compound 4b and MDM2 based on
its docking pose generated by Autodock, which is one of the most widely used
docking programmes [23]. As shown in Figure 4, benzene ring A and the propoxyl
groups on the 2- and 4-position (R & R ) of benzene ring B occupied the
3
5
hydrophobic pockets of Phe19, Trp23, and Leu26, respectively. By analysing the
binding modes of known inhibitors to MDM2, we concluded that hydrophobic
aromatic rings with halogen substituents favourably occupied the Trp23 pocket.
Additionally, given that the biphenyl scaffold is a privileged structure in medicinal
chemistry, we designed and synthesised a series of compounds with phenyl
substituted at the 2-position (R ) of benzene ring B (1f-3f, 1h and 2h).Given that there
3
were no substituents on the ring A, which likely made it vulnerable to metabolism,
compounds with substituents on ring A were synthesised to make them metabolically
stable and to investigate the SAR on ring A. Halogens play a vital role in drug
development, and around 25% of the “top 200 brand name drugs by retail dollar in
2
009” possesses halogens in their chemical structures. Additionally, halogens show a
potential ability to form halogen bonding with the target. Consequently, the
substituents chosen here were primarily halogens (F and Cl) [24]. In addition, other
compounds with various substituents on benzene ring B (1b-7b) were synthesised to
assay their binding affinity to MDM2 using fluorescence polarization assay.
2
.4 Chemistry
As shown in Scheme 1, compound 4b and several derivatives were synthesised.
, 4-Dihydroxybenzaldehyde was used as the starting material, and it was used in
2
reactions with alkyl iodide to yield compounds 1a-3a. These compounds were then
condensed with substituted indolin-2-one in ethanol under reflux using piperidine as
the base to afford the target compounds 1b-7b.
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The configuration of the double bond in compounds 1b-7b was assigned to E
based on the results of the NOESY experiment. In this experiment, a strong
interaction between the hydrogen atom at the 4-position of ring A and the 2-position
(
R
) of ring B was observed, and there was no interaction between the hydrogen atom
3
on the double bond and the hydrogen bond at the 4-position (R ) of ring A (scheme 1).
5
Additionally, the X-ray single crystal structure of compound 1b was determined. As
shown in Figure 5, benzene rings A and B were on the same side of the double bond,
as expected.
Based on the binding mode of compound 4b to MDM2, a series of derivatives
with substituted phenyl at the 2-position (R ) of benzene ring B were designed and
3
synthesised. As depicted in scheme 2, substituted bromobenzene was coupled with
bis(pinacolato)diboron using tetrakis(triphenylphosphine)palladium as the catalyst to
produce compounds 1d and 2d. Under the conditions of the Suzuki coupling reaction,
compound 2d was reacted with substituted 2-bromobenzaldehyde to yield compounds
1
e and 2e, which were further condensed with the substituted indolin-2-one to afford
the target compounds 1f-3f. However, when compound 1d was coupled with the
substituted 2-bromobenzaldehyde, no trace of the corresponding target compounds
was detected. We therefore adjusted the reaction sequence. The substituted
indolin-2-one was first condensed with 2-bromo-4-methoxybenzaldehyde to produce
compounds 1g and 2g, which were then coupled with compound 1d under the
conditions of the Suzuki coupling reaction to afford the desired compounds. The
configuration of the double bond of all the target compounds was confirmed using the
NOESY experiment.
2
.5 MDM2 Binding Affinity and structure activity relationships
The MDM2 binding affinity of all the target compounds was determined by
fluorescence polarization assay. Nutlin-3a was used as positive control to validate our
assay, and the results were summarized in table 1.
As shown in table 1, our assay was validated by the positive control nutlin-3a, of
7

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which the K value (0.11µM) was comparable to the reported one (0.23 µM). [12] In
i
general, all of the synthesized compounds showed high binding affinity to MDM2,
and this was especially true for compounds 1b, 2b, 1f, 2f, 1h and 2h, which exhibited
superior or comparable activity to nutlin-3a. Unexpectedly, of all the synthesized
compounds, 1b showed the most potent activity with a K value of 0.093 µM. This
i
effect was not consistent with the binding mode of 4b to MDM2 given that the Trp23
pocket was less occupied in the presence of the methoxyl group compared to its
n-propoxyl substituted counterpart. The only reasonable explanation is that compound
1
b displayed a distinct binding mode to MDM2. As depicted in Figure 6, a binding
mode of compound 1b to MDM2 was proposed based on its docking pose generated
by Autodock. A hydrogen bond was formed between His-96 of MDM2 and the
carbonyl group of compound 1b, and the ring A and B just occupied the Phe19 and
Trp23 pocket, respectively. The Leu26 pocket was less occupied.
In terms of the structure-activity relationships, introduction of the chloro group at
5
-position (R ) of the indolin-2-one ring (2b) had little influence on the binding
2
affinity compared to 1b. However, decreased inhibitory activity was observed when
replacing the H atom at 6-position (R ) of the indolin-2-one (1b) with a fluoro group
1
(
3b). Replacing the methoxyl group at 2-position (R ) of B ring (2b) with a bromine
3
atom (1g) also slightly decreased the activity. In the case of compound 4b, both
introduction of a chloro substituent at 5 or 6-position (R & R ) and substitution the
2
1
iso-propoxyl group for the n-propoxyl group did not alter the binding affinity. When
the n-propoxyl groups at the 2- and 4-positon (R & R ) of the B ring were replaced
3
5
by 4-chloro phenyl or 4-cyano phenyl substituents and a methoxyl group, respectively,
we observed an slight increase in the binding affinity to MDM2, which was in
consistence with the binding mode of compound 4b to MDM2. Migration of the
methoxyl group from 4-position (R ) to 3-position (R ) of ring B (3f) showed little
5
4
influence on the binding affinity.
The binding mode of compound 2h to MDM2 was also proposed based on its
docking pose generated by Autodock. As depicted in Figure 7, compound 2h shared a
8

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similar binding mode to that of compound 4b. As expected, the 4-chloro phenyl group
occupied the Trp-23 hydrophobic pocket, but the methoxyl group left the Phe-19
pocket less occupied, and this may explain why compounds with substituted phenyl at
ring B only showed slight increase in binding affinity compared to their propoxyl
substituted counterparts.
2
.6 In-vitro antiproliferative activity
The synthesized compounds were further assayed for their antiproliferative
activity in human colon cancer (HCT116 with wild type p53), HCT116 (p53 null),
lung adenocarcinoma (A549) and breast adenocarcinoma cells (MCF7) cells using
MTT assay with 48 h exposure time of the tested compounds, and nutlin-3a was used
as positive control. The results were summarized in table 2.
In general, all of the synthesized compounds showed a certain degree of
antiproliferative activity against HCT116 (both wild type p53 and p53 null) cells, and
the majority of the target compounds also presented some antiproliferative activity
against the A549 and MCF7 cell lines. The difference in cytotoxicity of synthesized
compounds between tumor cells with wild type p53 (HCT116, A549, MCF7) may be
attributed to: firstly, as the target, the amounts of endogenous MDM2 in three cell
lines are different, so the cytotoxicity of selected compounds to the three cancer cell
lines varied; secondly, the three cell lines are from different tissues; last but not least,
other tumor suppressor genes may be present in A549 and MCF-7, which may lead
to reduced activity. Notably, compounds 1b, 3b-7b, 1f, 2f, 1h and 2h exhibited
superior or comparable activity against HCT116 (p53 +/+) to nutlin-3a. In particular,
compound 1b with the most potent inhibitory activity against MDM2-p53 interaction
(
K = 0.093 µM) also showed the best inhibitory activity against HCT116 (wild type
i
p53). Additionally, all the tested compounds showed relatively lower GI50 values
against HCT116 (wild type p53) compared to HCT116 with p53 deleted,
demonstrating the selectivity of the synthesized compounds over cancer cell lines
with deleted p53. Since compound 1b showed both the highest binding affinity to
9

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MDM2 and the most potent antiproliferative activity, it was chosen for further
mechanism study.
2
.4 Western blotting analyses
p53 is activated by the inhibition of the MDM2-p53 interaction, resulting in the
increased expression of wild-type p53 in cells. Subsequently, p53 activation results in
increased levels of p21 and MDM2. [7] To further verify the anticancer mechanism of
this class of compounds, western blotting was used to assess the effects of compound
1
b in HCT116 cells. The cells were treated with compound 1b or the positive control
nutlin-3a for 48 h. Next, the cells were lysed and protein extracts were analysed by
western blotting to determine the levels of p53, p21 and MDM2. As shown in Figure
8
, compound 1b increased the levels of p21, p53 and MDM2 in a dose-dependent
manner in HCT116 cells.
2
.5 Cell cycle analysis
To gain a better understanding of the mechanisms that underlie the
antiproliferative activities of this series of compounds, we investigated the effect of
compound 1b on the cell cycle in HCT116 (wild type p53) cells. HCT116 cells were
treated with compounds 1b and nutlin-3a for 48 h. The fixed cells were stained with
propidium iodide (PI) and detected using flow cytometry. As shown in Figure 9, both
compound 1b and nutlin-3a arrested cells in G2/M phase and induced apoptosis in a
dose-dependent manner in HCT116 cells. Moreover, at the same concentrations,
compound 1b induced more apoptosis than nutlin-3a in HCT116 cells.
2
.6 In vivo antitumor activity
To further test the therapeutic potential for the synthesized compounds,
compound 1b was chosen to evaluate its antitumor activity in the BALB/c mice
bearing colon carcinoma cell line CT26 (CT26 rather than HCT116 cell line was
chosen to evaluate the antitumor activity of 1b preliminarily, and the HCT116
1
0

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xenograft model will be employed to further investigate the in-vivo antitumor activity
of 1b in near future). Before the experiment, the configuration of double bonds of
compound 1b was confirmed again by NOE experiment, and the isomerization of the
double bond was not observed.
First, compound 1b was dissolved in 10% DMSO and administered via an
intraperitoneal injection to two groups of BALB/c mice (10 mice for each group) at
doses of 500 mg/kg and 1000 mg/kg, respectively, to determine the maximum
administration dosage. No death or visible side effects were observed in the 500
mg/kg dosage group over 8 days. However, all of the mice died within 24 h after the
injection of 1000 mg/kg compound 1b. Therefore, the doses used to evaluate the
efficacy of compound 1b were initially set at 50 mg/kg and 100 mg/kg, and
oxaliplatin (1.5 mg/kg) was used as the positive control.
CT26 cancer cell suspensions were implanted subcutaneously into the right axilla
region of the mice, and all the mice were divided randomly into the following 5
groups (10 mice/group): compound 1b (50 mg/kg and 100 mg/kg), oxaliplatin (1.5
mg/kg), vehicle and control groups. The BALB/c mice bearing CT26 colon carcinoma
were treated with compound 1b (100 and 50 mg/kg), oxaliplatin (1.5 mg/kg) or the
vehicle via an intraperitoneal injection, and the each treatment was given once daily
for 10 days. At the end of the study, all of the mice were weighed and sacrificed, and
the tumor s were excised and weighed for analysis. The ratio of tumor growth
inhibition was calculated using the following formula: (A–B)/A×100% (where A is
the average tumor weight of the control group, and B is the average tumor weight of
the experimental group). The results were summarised in Table 3.
As shown in Table 3, compound 1b effectively inhibited tumor growth in a
dose-dependent manner, with a tumor growth inhibition (TGI) of 18.1% and 32.4%
for the low and high-dose group, respectively. The TGIs observed for compound 1b
were inferior to those for the positive control, oxaliplatin, but were superior to
oxaliplatin in terms of drug tolerance, as determined by the difference in body weight
before and after treatment. The body weight of the oxaliplatin group decreased by
11

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4
.01 g, whereas it only decreased by 1.02 and 0.74 g for the high- and low-dose group
of 1b, respectively.
3
. Conclusion
In this study, an integrated virtual screening strategy combining both
pharmacophore- and structure-based approaches identified a hit compound with a
E)-3-benzylideneindolin-2-one scaffold as novel inhibitor of the MDM2-p53
(
interaction. Further optimization led to several derivatives with potent inhibitory
activity, among which 1b showed both the highest binding affinity to MDM2 and the
most potent antiproliferative activity against HCT116 (wild type p53). Additionally, it
possesses a K value of 0.093 µM with only 21 non-hydrogen atoms, which means it
i
possesses the highest ligand efficiency value (LE = 1.4 pIC /N non-hydrogen atoms) [25, 26]
5
0
among all the synthesized compounds. Consequently, compound 1b represents
promising lead compound for the development of novel anticancer drugs as
MDM2-p53 inhibitors and shows potential for optimization to improve its activity
without sacrificing its druggability. The detailed structural optimisation based on its
binding mode to MDM2 proposed in this work is being carried out in our laboratory.
4
. Experimental section
5
.1 Synthesis and characterisation
1
13
H and C NMR spectra were recorded using TMS as the internal standard in
DMSO-d or CDCl with a Bruker BioSpin GmbH spectrometer at 400 and 600 MHz,
6
3
respectively. The mass spectra (MS) were recorded on a Thermo Scientific LTQ
ORBITRAP instrument with an ESI mass selective detector. Melting points (m.p.)
were determined using an SRS-OptiMelt automated melting point instrument, without
correction. Flash column chromatography was performed with silica gel (200-300
mesh) purchased from Qingdao Haiyang Chemical Co. Ltd.
5
.1.1 General procedure A for the preparation of 1a-3a
2
, 4-Dihydroxybenzaldehyde (200 mmol) and alkylation agent (440 mmol) were
1
2

ACCEPTED MANUSCRIPT
dissolved in 200 ml anhydrous DMF, and potassium carbonate (600 mmol) was added.
The mixture was heated at 85 °C for 3.5 h, and TLC analysis indicated that the
reaction was complete. The mixture was filtered, and the filtrate was concentrated in
vacuo. The resulting residue was slowly dissolved in ethyl acetate, and was rapidly
washed with distilled water. The organic layer was then dried over anhydrous MgSO4
for 2 h and evaporated. The residue was recrystallised using anhydrous ethanol to give
the corresponding product.
5
.1.1.1 2,4-Dimethoxy-benzaldehyde (1a)
2
, 4-Dihydroxybenzaldehyde and Methyl iodide were dissolved in DMF, and the
mixture was treated with K CO according to general procedure A to afford the target
2
3
compound as a white solid with the following characteristics: 95% yield; m.p.
1
6
1
3
9.1-71.2 °C; H NMR (600 MHz, DMSO-d ): δ 10.16 (s, 1H), 7.64 (d, J = 8.4 Hz,
6
H), 6.66 (d, J = 2.4 Hz, 1H), 6.62 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 3.89 (s, 3H), 3.86 (s,
+
H); MS (ESI): [M+H] m/z 167.1.
5
.1.1.2 2,4-Dipropoxy-benzaldehyde (2a)
2
, 4-Dihydroxybenzaldehyde and 1-bromopropane were dissolved in DMF, and the
mixture was treated with K CO according to general procedure A to afford the target
2
3
1
compound as a pale yellow solid with the following characteristics: 90% yield; H
NMR (600 MHz, DMSO-d ): δ 10.33 (s, 1H), 7.79 (d, J = 9.0 Hz, 1H), 7.12 (dd, J =
6
1
.8 Hz, 9.0 Hz, 1H), 6.42 (d, J = 1.8 Hz, 1H), 3.96-4.01 (m, 4H), 1.80-1.88 (m, 4H),
+
1
.03-1.07 (m, 6H); MS (ESI): [M+H] m/z 223.3.
5
.1.1.3 2, 4-Diisopropoxy-benzaldehyde (3a)
2
, 4-Dihydroxybenzaldehyde and 2-bromopropane were dissolved in DMF, and the
mixture was treated with K CO according to general procedure A to afford the target
2
3
1
compounds as a colourless oil with the following characteristics: 90% yield; H NMR
600 MHz, DMSO-d ): δ 10.17 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H), 6.61 (d, J = 2.4 Hz,
(
6
1
H), 6.56 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 4.72-4.78 (m, 2H), 1.26-1.31 (m, 12H); MS
1
3

ACCEPTED MANUSCRIPT
+
(
ESI): [M+H] m/z 223.3.
5
.1.2 General procedure B for the preparation of 1b-7b, 1f-3f, and 1g-2g
Piperidine (14.4 mL) was slowly added to a suspension of indolone (70 mmol)
and substituted aldehyde (84 mmol) in 80 mL ethanol. The mixture was heated under
reflux for 1.5 h, and TLC analysis indicated when the reaction was complete. After
concentration in vacuo, the resulting residue was redissolved in ethyl acetate. Next,
the mixture was successively washed with water and brine. The organic layer was
dried over anhydrous MgSO , filtered and concentrated, and the resulting residue was
4
recrystallised from anhydrous ethanol to give the corresponding product.
5
.1.2.1 3-(2,4-Dimethoxy-benzylidene)-1,3-dihydro-indol-2-one (1b)
,4-Dimethoxy-benzaldehyde and 1,3-dihydro-indol-2-one were dissolved in
2
ethanol, and the mixture was treated with piperidine, according to general procedure
B to afford (1b) as a yellow solid with the following characteristics: 88% yield; m.p.
1
2
7
6
06.4-208.1 °C; H NMR (600 MHz, CDCl ): δ 7.95 (s, 1H), 7.73(d, J = 8.4 Hz, 1H),
3
.66 (d, J = 8.4 Hz, 1H), 7.55 (br, 1H), 7.18 (t, J = 8.4 Hz, 1H), 6.85-6.89 (m, 2H),
.53 (dd, J = 1.8 Hz, 8.4 Hz, 1H), 6.52 (d, J = 1.8 Hz, 1H), 3.90 (s, 3H), 3.87 (s, 3H);
1
3
C NMR (100 MHz, DMSO-d ): δ 168.93(C), 162.70(C), 159.42(C), 142.44(C),
6
1
1
5
2
31.71(C), 130.66(C), 129.44(CH), 125.21(CH), 121.95(CH), 121.37(CH),
20.99(CH), 115.31(C), 109.93(CH), 105.32(CH), 98.51(CH), 55.81(CH3),
+
5.53(CH ); HRMS (ESI): Calculated for [M+H] (C H N O ), requires m/z
3
17 16
1
3
82.1130, found 282.1116.
5
.1.2.2 5-Chloro-3-(2,4-dimethoxy-benzylidene)-1,3-dihydro-indol-2-one (2b)
,4-Dimethoxy-benzaldehyde and 5-chloro-1,3-dihydro-indol-2-one were
2
dissolved in ethanol, and the mixture was treated with piperidine according to general
procedure B to afford (2b) as a yellow solid with the following characteristics: 86%
1
yield; m.p. 238.3-239.6 °C; H NMR (600 MHz, CDCl ): δ 8.01 (s, 1H), 7.68 (br, 1H),
3
1
4

ACCEPTED MANUSCRIPT
7
.62 (d, J = 1.8 Hz, 1H), 7.16 (dd, J = 1.8 Hz, 8.4 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H),
.61 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H);
6
1
3
C NMR (100 MHz, DMSO-d ): δ 168.56 (C), 163.11 (C), 159.37 (C), 141.16
6
(CH), 133.65 (C), 130.83 (C), 128.92 (CH), 124.80 (C), 124.23 (CH), 122.80 (C),
1
21.21 (CH), 114.92 (CH), 111.30 (CH), 105.54 (C), 98.62 (CH), 55.80 (CH ), 55.65
3
+
(
CH ); HRMS (ESI): Calculated for [M+H] (C H Cl N O ), requires m/z 316.0741,
3
17 15
1
1
3
found 316.0738.
5
.1.2.3 3-(2,4-Dimethoxy-benzylidene)-6-fluoro-1,3-dihydro-indol-2-one (3b)
,4-Dimethoxy-benzaldehyde and 6-fluoro-1,3-dihydro-indol-2-one were
2
dissolved in ethanol, and the mixture was treated with piperidine according to general
procedure B to afford 3b as a yellow solid with the following characteristics: 86%
1
yield; m.p. 226.3-227.6 °C; H NMR (600 MHz, CDCl ): δ 8.01 (s, 1H), 7.86 (br, 1H),
3
7
6
3
1
.67 (d, J = 9.0 Hz, 1H), 7.37 (dd, J = 2.4 Hz, 9.0 Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H),
.79 (d, J = 8.4 Hz, 1H), 6.59 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 6.52 (d, J = 2.4 Hz, 1H),
13
.90 (s, 3H), 3.87 (s, 3H); C NMR (100 MHz, DMSO-d ): δ 168.91(C), 163.07(C),
6
59.55(C), 158.34(C), 138.76(C), 133.38(C), 130.75(C), 124.92(CH), 122.36(CH),
116.06(CH), 114.92(C), 110.60(CH), 105.50(CH), 98.64(CH), 97.61(CH),
+
5
5.87(CH ), 55.66(CH ); HRMS (ESI): Calculated for [M+H] (C H F N O ),
3 3 17 15 1 1 3
requires m/z 300.0958, found 300.1036.
5
.1.2.4 3-(2,4-Dipropoxy-benzylidene)-1,3-dihydro-indol-2-one (4b)
, 4-Dipropoxy-benzaldehyde and 1, 3-dihydro-indol-2-one were dissolved in
2
ethanol, and the mixture was treated with piperidine according to general procedure B
to afford 4b as a yellow solid with the following characteristics: 86% yield; m.p.
1
1
7
8
1
62.3-163.4 °C; H NMR (600 MHz, CDCl ): δ 7.99 (s, 1H), 7.72 (d, J = 7.8 Hz, 1H),
3
.69 (d, J = 7.8 Hz, 1H),7.18 (d, J = 8.4 Hz, 1H ), 6.87 (m, 2H), 6.54 (dd, J = 2.4 Hz,
.4 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 4.00 (t, J = 6.6 Hz, 2H), 3.97 (t, J = 6.6 Hz, 2H),
1
3
.87-1.81 (m, 4H), 1.75 (t, J = 7.2 Hz, 3H), 1.02 (t, J = 7.2 Hz, 3H); C NMR (100
1
5

ACCEPTED MANUSCRIPT
MHz, DMSO-d ): δ 168.98(C), 162.09(C), 158.85(C), 142.36(C), 131.79(C),
6
1
30.73(C), 129.35(CH), 125.04(CH), 121.93(CH), 121.44(CH), 120.94(CH),
115.34(C), 109.88(CH), 105.75(CH), 99.49(CH), 69.53(CH3), 69.30(CH2),
+
2
2.03(CH ), 21.96(CH ), 10.44(CH ); HRMS (ESI): Calculated for [M+H]
2 2 3
(
C H N O ) requires m/z 338.1756, found 338.1761.
21 23 1 3
5
.1.2.5 5-Chloro-3-(2,4-dipropoxy-benzylidene)-1,3-dihydro-indol-2-one (5b)
,4-Dipropoxy-benzaldehyde and 5-chloro-1,3-dihydro-indol-2-one were
2
dissolved in ethanol, and the mixture was treated with piperidine according to general
procedure B to afford 5b as a yellow solid with the following characteristics: 86%
1
yield; m.p. 171.1-172.2 °C; H NMR (600 MHz, CDCl ): δ 8.03 (s, 1H), 7.65 (d, J =
3
8
.4 Hz, 1H), 7.63 (d, J = 2.4 Hz, 1H ), 7.15 (d, J = 2.4 Hz, 8.4 Hz, 1H), 6.82(d, J = 8.4
Hz, 1H ) 6.58 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 3.97-4.02 (m,
13
4
H), 1.79-1.88 (m, 4H), 1.10 (t, J = 7.8 Hz, 3H), 1.02 (t, J = 7.2 Hz, 3H). C NMR
(
100 MHz, DMSO-d ): δ 168.69(C), 162.55(C), 159.00(C), 141.10(C), 133.70(C),
6
1
30.95(C), 128.80(C), 124.81(CH), 124.20(CH), 123.19(CH), 121.51(CH),
114.94(CH), 111.22(CH), 105.93(C), 99.57(CH), 69.61(CH2), 69.40(CH2),
+
2
2.05(CH ), 21.94(CH ), 10.44(CH ); HRMS (ESI): Calculated for [M+H]
2 2 3
(
C H Cl N O ), requires m/z 372.1367, found 372.1354.
21 23 1 1 3
5
.1.2.6 5-Chloro-3-(2,4-diisopropoxy-benzylidene)-1,3-dihydro-indol-2-one (6b)
,4-Dipropoxy-benzaldehyde and 5-chloro-1,3-dihydro-indol-2-one were
2
dissolved in ethanol, and the mixture was treated with piperidine according to general
procedure B to afford 6b as a yellow solid with the following characteristics: 86%
1
yield; m.p. 170.8-172.1 °C; H NMR (600 MHz, CDCl ): δ 8.05 (s, 1H), 8.01 (s, 1H),
3
7
6
4
.66 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 2.4 Hz, 1H), 7.13 (dd, J = 2.4 Hz, 8.4 Hz, 1H),
.76 (d, J = 8.4 Hz, 1H) 6.52 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 6.42 (d, J = 2.4 Hz, 1H),
13
.58-4.67 (m, 2H), 1.40 (d, J = 6.0 Hz, 6H), 1.36 (d, J = 6.6 Hz, 6H); C NMR: (100
MHz, DMSO-d ): δ 168.75(C), 161.34(C), 158.09(C), 141.07(C), 134.14(C),
6
1
6

ACCEPTED MANUSCRIPT
1
31.09(C), 128.73(C), 124.76(CH), 123.94(CH), 123.18(CH), 121.48(CH),
115.42(CH), 111.20(C), 106.80(CH), 101.46(CH), 70.41(CH), 69.67(CH),
+
2
3
1.83(CH ); HRMS (ESI): Calculated for [M+H] (C H Cl N O ) requires m/z
3
21 23
1
1
3
72.1367, found 372.1385.
5
.1.2.7 6-Chloro-3-(2,4-diisopropoxy-benzylidene)-1,3-dihydro-indol-2-one (7b)
,4-Diisopropoxy-benzaldehyde and 6-chloro-1,3-dihydro-indol-2-one were
2
dissolved in ethanol, and the mixture was treated by piperidine according to general
procedure B to afford 7b as a yellow solid with the following characteristics: 86%
1
yield; m.p. 173.1-173.9 °C; H NMR (600 MHz, CDCl ): δ 7.91 (s, 1H), 7.62 (d, J =
3
8
8
2
.4 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 6.90 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 6.85 (d, J =
.4 Hz, 1H), 6.78 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 4.71-4.75 (m,
1
3
H), 1.33 (d, J = 6.0 Hz, 6H), 1.28 (d, J = 6.6 Hz, 6H); C NMR (100 MHz,
DMSO-d ): δ 169.01(C), 161.17(C), 158.08(C), 143.63(C), 133.13(C), 131.01(C),
6
1
1
23.59(C), 123.21(CH), 120.71(CH), 120.42(CH), 115.60(C), 109.80(CH),
06.83(CH), 101.47(CH), 70.35(CH), 69.58(CH), 21.48(CH ); HRMS (ESI):
3
+
Calculated for [M+H] (C H Cl N O ) requires m/z 372.1367, found 372.1352.
21
23
1
1
3
5
.1.2.8 2'-(5-Chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-5'-methoxy-biphenyl
4-carbonitrile (1f)
'-Formyl-5'-methoxy-biphenyl-4-carbonitrile and 6-chloro-1,3-dihydro-indol-2-
-
2
one were dissolved in ethanol, and the mixture was treated with piperidine according
to general procedure B to afford 1f as a yellow solid with the following characteristics:
1
8
7
1
2
6% yield; m.p. >280°C; H NMR (600 MHz, CDCl ): δ 7.81 (d, J = 8.4 Hz, 2H),
3
.70 (d, J = 8.4 Hz, 2H), 7.60 (s, 1H), 7.59 (d, J = 1.8 Hz, 1H), 7.46 (d, J = 8.4 Hz,
H), 7.21 (dd, J = 1.8 Hz, 8.4 Hz, 1H), 7.07 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.01 (d, J =
1
3
.4 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 3.95 (s, 3H); C NMR: (100 MHz, DMSO-d ):
6
δ 168.80(C), 168.16(C), 144.10(C), 142.23(C), 141.50(C), 136.56(C), 132.35(C),
31.19(CH), 130.84(CH), 130.52(CH), 129.39(CH), 128.19(C), 126.21(C),
1
1
7

ACCEPTED MANUSCRIPT
1
24.93(CH), 124.30(CH), 122.85(CH), 121.82(CH), 118.56(CH), 115.40(C),
1
14.44(CH), 111.45(CH), 110.80(CH), 55.69(CH ); HRMS (ESI): Calculated for
3
+
[
M+H] (C H Cl N O ) requires m/z 387.0900, found 387.0895.
23 15 1 2 2
5
.1.2.9 2'-(6-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-5'-methoxy-biphenyl
-4-carbonitrile (2f)
2'-Formyl-5'-methoxy-biphenyl-4-carbonitrile and 6-fluoro-1,3-dihydro–indol
-2-one were dissolved in ethanol, and the mixture was treated with piperidine
according to general procedure B to afford 2f as a yellow solid with the following
1
characteristics: 86% yield; m.p. >280°C; H NMR (600 MHz, CDCl ): δ 7.91 (s, 1H),
3
7
.70 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 2.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.46 (d, J
8.4 Hz, 2H), 7.21 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.07 (dd, J = 1.8 Hz, 8.4 Hz, 1H),
=
1
3
7
.01 (d, J = 2.4 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 3.88 (s, 3H); C NMR (100 MHz,
DMSO-d ): δ 168.43(C), 160.71(C), 158.39(C), 155.75(C), 144.12(C), 142.14(C),
6
1
1
39.07(C), 136.34(CH), 132.36(CH), 131.14(CH), 130.51(CH), 126.79(CH),
24.27(CH), 122.13(C), 118.66(CH), 116.30(CH), 116.07(C), 115.46(CH),
1
14.41(CH), 110.79(C), 109.53(CH), 109.26(CH), 55.68(CH ); HRMS (ESI):
3
+
Calculated for [M+H] (C H F N O ) requires m/z 371.1196, found 371.1201.
23
15
1
2
2
5
.1.2.10 2'-(6-Chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-6'-methoxy-biphenyl-
4
-carbonitrile (3f)
2'-Formyl-6'-methoxy-biphenyl-4-carbonitrile and 6-chloro-1,3-dihydro–indol
-2-one were dissolved in ethanol, and the mixture was treated with piperidine
according to general procedure B to afford 3f as a yellow solid with the following
1
characteristics: 86% yield; m.p. >280°C; H NMR (400 MHz, DMSO-d ): δ 10.72 (s,
6
1
H), 7.94 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.0 Hz, 1H), 7.42
(
(
d, J = 2.0 Hz, 1H), 7.29-7.32 (m, 1H), 7.23 (dd, J = 2.0 Hz, 8.0 Hz, 1H), 7.17-7.19
13
m, 1H), 6.90 (d, J = 8.0 Hz, 1H), 3.92 (s, 3H); C NMR: (100 MHz, DMSO-d ): δ
6
1
68.61(C), 166.88(C), 143.13(C), 141.50(C), 139.41(C), 136.52(C), 135.32(C),
1
8

ACCEPTED MANUSCRIPT
1
1
35.07(C), 129.46(CH), 128.51(CH), 128.07(C), 124.50(CH), 124.43(CH),
22.62(CH), 122.31(CH), 121.64(CH), 113.90(C), 113.52(CH), 112.53(CH),
+
110.77(CH), 110.09(CH), 57.04(CH). HRMS (ESI): Calculated for [M+H]
(
C H Cl N O ) requires m/z 387.0900, found 387.0890.
2
3
15
1
2
2
5
.1.2.11 3-(2-Bromo-4-methoxy-benzylidene)-5-chloro-1,3-dihydro-indol-2-one (1g)
-Bromo-4-methoxy-benzaldehyde and 6-chloro-1,3-dihydro–indol-2-one were
2
dissolved in ethanol, and the mixture was treated with piperidine according to general
procedure B to afford 1g as a yellow solid with the following characteristics: 74%
1
yield; m.p. 247.5-249.2 °C; H NMR (600 MHz, CDCl ): δ 7.82 (s, 1H), 7.70 (br, 1H),
3
7
.65 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 2.4 Hz, 1H), 7.25-7.26 (m, 1H), 7.18 (d, J = 2.4
1
3
Hz, 1H), 6.97 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 3.89 (s, 3H); C
NMR (100 MHz, DMSO-d ): δ 168.07(C), 161.26(C), 141.73(C), 135.65(C),
6
1
1
31.40(C), 129.83(C), 126.95(C), 126.02(CH), 125.05(CH), 124.93(CH), 122.36(CH),
21.82(C), 118.22(CH), 114.23(CH), 111.66(CH), 56.02(CH ); HRMS (ESI):
3
+
Calculated for [M+H] (C H Br Cl N O ) requires m/z 363.9740, found 363.9725.
16
12
1
1
1
2
5
.1.2.12 3-(2-Bromo-4-methoxy-benzylidene)-5-fluoro-1,3-dihydro-indol-2-one (2g)
-Bromo-4-methoxy-benzaldehyde and 5-fluoro-1,3-dihydro-indol-2-one were
2
dissolved in ethanol, and the mixture was treated with piperidine according to general
procedure B to afford 2g as a yellow solid with the following characteristics: 92%
1
yield; m.p. 273.7-274.2 °C; H NMR (600 MHz, CDCl ): δ 7.84 (s, 1H), 7.65 (d, J =
3
8
.4 Hz, 1H), 7.41 (d, J = 1.8 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.19 (dd, J = 1.8 Hz,
.4 Hz, 1H), 6.97 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 6.80 (d, J = 2.4 Hz, 1H), 3.88 (s, 3H);
8
1
3
C NMR (100 MHz, DMSO-d ): δ 168.61(C), 156.60(C), 144.91(C), 136.52(C),
6
1
1
35.07(C), 129.46(C), 128.51(C), 124.50(CH), 124.43(CH), 122.62(CH), 122.31(CH),
+
21.64(C), 113.52(CH), 112.53(CH), 110.09(CH), 57.04(CH ); MS (ESI): [M+H]
3
m/z 350.0.
1
9

ACCEPTED MANUSCRIPT
5
.1.3 General procedure C of preparation of 1d and 2d
Substituted bromobenzene (80 mmol), bis(pinacolato)diboron (120 mmol),
potassium acetate (160 mmol) and triphenylphosphine palladium (8 mmol) were
dissolved in anhydrous DMSO under N protection. The mixture was heated to 85°C
2
for 4 h, and TLC analysis indicated when the reaction was complete. Next, distilled
water was added. The mixture was extracted by ethyl acetate, dried over anhydrous
MgSO for 2 h and evaporated. The residue was chromatographed on silica gel and
4
eluted with Petroleum ether/Ethyl acetate to produce 1d and 2d.
5
.1.3.1 2-(4-Chloro-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (1d)
-Bromo-4-chloro-benzene was treated with bis(pinacolato)diboron according to
1
general procedure C to afford 1d as a white solid with the following characteristics:
1
6
2
0% yield; m.p. 50.1-51.1 °C; H NMR (600 MHz, DMSO-d ): δ 7.68 (d, J = 8.4 Hz,
6
+
H), 7.46 (d, J = 8.4 Hz, 2H), 1.28 (s, 12H); MS (ESI): [M+H] m/z 239.1.
5
.1.3.2 4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzonitrile (2d)
The 4-bromo-benzonitrile was treated with bis(pinacolato)diboron according to
general procedure C to afford 2d as a white solid with the following characteristics:
1
7
2
4% yield; m.p. 93.5-94.3°C; H NMR (400 MHz, DMSO-d ): δ 7.66 (d, J = 8.4 Hz,
6
+
H), 7.43 (d, J = 8.4 Hz, 2H), 1.30 (s, 12H); MS (ESI): [M+H] m/z 230.1.
5
.1.4.1 2'-Formyl-5'-methoxy-biphenyl-4-carbonitrile (1e)
Caesium carbonate (350 mmol) was added to a stirred suspension of 1d (150
mmol) and 2-bromo-4-methoxy–benzaldehyde (150 mmol) in 100 mL DMF, followed
by the addition of triphenylphosphine palladium (15 mmol). The reaction was heated
at 120 °C for 4 h and then allowed to cool to room temperature. The organic solvent
was removed in vacuo and diluted in ethyl acetate. The entire mixture was washed
with ice water (200 mL). The organic layer was then dried over anhydrous MgSO4.
The organic solvent was removed by rotary evaporation, and the residue was purified
2
0

ACCEPTED MANUSCRIPT
using column chromatography with petroleum ether/ethyl acetate to afford 1e with the
1
following characteristics: 70% yield; m.p. 138.7-139.9 °C; H NMR (600 MHz,
CDCl ): δ 10.21 (s, 1H), 7.83 (d, J = 9.0 Hz, 2H), 7.50 (d, J = 9.0 Hz, 2H), 7.38 (d, J
3
=
2.4 Hz, 1H), 6.77 (dd, J = 2.4 Hz, 9.0 Hz, 1H), 6.64 (d, J = 9.0 Hz, 1H), 3.84 (s, 3H);
+
MS (ESI): [M+H] m/z 238.1.
5
.1.4.2 2'-Formyl-6'-methoxy-biphenyl-4-carbonitrile (2e)
Caesium carbonate (350 mmol) was added to a stirred suspension of 1d (150
mmol) and 2-bromo-3-methoxy–benzaldehyde (150 mmol) in 100 mL DMF, followed
by the addition of triphenylphosphine palladium (15 mmol). The reaction was heated
at 120 °C for 5.5 h and then allowed to cool to room temperature. The organic solvent
was removed in vacuo and diluted in ethyl acetate. The entire mixture was washed
with ice water (200 mL). The organic layer was then dried over anhydrous MgSO4.
The organic solvent was removed by rotary evaporation, and the residue was purified
using column chromatography with petroleum ether/ethyl acetate to afford 1e with the
1
following characteristics: 55% yield; m.p. 132.7-134.2°C; H NMR (600 MHz,
DMSO-d ): δ 9.61 (s, 1H), 7.90 (d, J = 7.8 Hz, 2H), 7.60 (t, J = 7.8 Hz, 1H), 7.52 (d, J
6
+
=
7.8 Hz, 2H), 7.46 (d, J = 7.8 Hz, 2H), 3.75 (s, 3H); MS (ESI): [M+H] m/z 238.1.
5
.1.5 General procedure C for the preparation of 1h and 2h
Caesium carbonate (300 mmol) was added to a stirred suspension of 1g or 2g
(100 mmol) and 1d in 100 mL DMF, followed by the addition of triphenylphosphine
palladium (10 mmol). The reaction was heated at 120°C for 4 h and then allowed to
cool to room temperature. The organic solvent was removed in vacuo and diluted in
ethyl acetate. The entire mixture was washed with ice water (200 mL). The organic
layer was then dried over anhydrous MgSO . The organic solvent was removed by
4
rotary evaporation, and the residue was purified using column chromatography with
petroleum ether/ethyl acetate to produce 1h and 2h.
2
1

ACCEPTED MANUSCRIPT
5
2
.1.5.1 5-Chloro-3-(4'-chloro-5-methoxy-biphenyl-2-ylmethylene)-1,3-dihydro-indol-
-one (1h)
1
g and 1d were dissolved in DMF, according to general procedure C to afford
1
compound 1h with the following characteristics: 54% yield; m.p. 202.6-204.1 °C; H
NMR (600 MHz, CDCl ): δ 7.80 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.60(d,
3
J = 3.0 Hz, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.20-7.22 (m, 1H), 7.07(d, J = 3.0 Hz, 1H),
1
3
7
.01 (dd, J = 3.0 Hz, 9.0 Hz, 1H), 6.81 (d, J = 9.0 Hz, 1H), 3.95 (s, 3H); C NMR
(
100 MHz, DMSO-d ): δ 168.08(C), 160.74(C), 144.05(C), 142.15(C), 141.46(C),
6
1
1
36.47(C), 132.26(C), 131.11(CH), 130.45(CH), 129.32(C), 126.20(C), 124.89(CH),
24.27(CH), 122.80(CH), 121.78(CH), 118.57(CH), 115.35(CH), 114.38(CH),
+
1
11.38(CH), 110.75(CH), 55.62(CH ); HRMS (ESI): Calculated for [M+H]
3
(
C H N O Cl ) requires m/z 395.0480, found 395.0429.
2
2
15
1
2
2
5
2
.1.5.2 3-(4'-Chloro-5-methoxy-biphenyl-2-ylmethylene)-5-fluoro-1,3-dihydro-indol-
-one (2h)
2g and 1d were dissolved in DMF according to general procedure C to afford
1
compound (2h) with the following characteristics: 65% yield; m.p. 268.2-269.7°C; H
NMR (600 MHz, CDCl ): δ 7.79 (s, 1H), 7. 78 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz,
3
2
H), 7.27 (d, J = 9.0 Hz, 1H), 7.26 (s, 1H) 7.02 (d, J = 9.0 Hz, 1H), 6.93 (d, J = 8.4
1
3
Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H), 6.82 (dd, J = 2.4 Hz, 8.4 Hz, 1H ) 3.92 (s, 3H); C
NMR (100 MHz, DMSO-d ): δ 168.09(C), 160.74(C), 144.06(C), 142.16(C),
6
1
41.47(C), 136.47(C), 132.26(C), 131.11(CH), 130.44(CH), 129.32(CH), 126.21(C),
24.88(CH), 124.28(CH), 122.80(C), 121.79(CH), 118.58(CH), 115.36(CH),
1
1
14.38(CH), 111.38(CH), 110.76(CH), 55.63(CH ); HRMS (ESI): Calculated for
3
+
[
M+H] (C H N O Cl F ) requires m/z 380.0775, found 380.0849.
22 15 1 2 1 1
5
.2 Biological assay
A549, HCT116 and MCF7 cell lines were purchased from the American Type
Culture Collection. MDM2 binding domain (1-118) was purchased from Creative
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2

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Biomart, and the PMDM-F was obtained from AnaSpec Inc. The positive control drug,
nutlin-3a, was purchased from Haoyuan Chempress Shanghai. All the tested
compounds were dissolved in DMSO to 100 mM stock solutions, and they were
further diluted with culture medium prior to use.
5
.2.1 MTT assay
The cells were seeded into a 96-well plate at a density of 5000 (100 ꢀL) per well
for 24 h, followed by drug treatment (100 ꢀL) for 48 h. Next, 20 ꢀL of 5 mg/ml MTT
was added to the medium, and the cells were incubated for 4 h at 37 °C and 5% CO2.
After removing the culture medium, 150 ꢀL of DMSO was added. The plates were
read using an enzyme-linked immunosorbent assay plate reader at 570 nm. The
viability of untreated cells was set as 100%, and the viability in the other groups was
calculated by comparing the optical density reading with the control. The GI values
5
0
were calculated using nonlinear regression analysis. The assays were repeated three
times.
5
.2.2 MDM2 binding assay
The dose-dependent binding experiments were carried out with serial dilution in
DMSO of compounds. A 3 ꢀL sample of the tested compounds and preincubated (for
3
0 min) MDM2 binding domain (1-118) (30 nM) and PMDM-F peptide (10 nM) in
the assay buffer (100 mM potassium phosphate, pH 7.5; 100 mg/mL bovine gamma
globulin; 0.02% sodium azide) was added into black 96-well microplates with
F-bottom and chimney wells (Corning) to produce a final volume of 115 ꢀL. For each
assay, the controls included the MDM2 binding domain and PMDM-F. The
polarization values were measured at room temperature using Biotek Synergy H2
with a 485 nm excitation filter, a 528 nm static and polarized filter. The K value was
i
calculated according to a reported method. [27]
5
.2.3 Western Blotting
HCT116 cells (wild type p53) were treated with various concentrations of the
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3

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indicated compound for 48 h. Next, the cells were harvested by centrifugation at 1000
g for 5 min. The cell pellets were washed with PBS, resuspended in lysis buffer (150
mM NaCl, 50 mM Tris (pH 8.0), 0.02% NaN , 0.01% PMSF, 0.2% Aprotinin, and 1%
3
TritonX-100, supplemented with protease inhibitor cocktail (Thermo Scientific)), and
centrifuged at 12000 g for 10 min. The total protein concentration was determined
using the Bio-Rad protein assay. The proteins were resolved by SDS-PAGE and
transferred to a polyvinylidene fluoride membrane. After blocking with 5% non-fat
milk in blocking buffer (PBS containing 0.1% Tween 20, pH 7.5), the membrane was
incubated with the indicated primary antibody for 2 h at room temperature and then
incubated with the appropriate peroxidase-conjugated secondary antibody. The
immunoreactive bands were visualised using the ECL Plus Western Blotting
Detection System (Piscataway, New Jersey, USA). Beta-actin was used as a loading
control.
5
.2.4 Cell Cycle Analysis
HCT116 cells were seeded at 2×10 cells/well in 6-well plates and cultured for 48
5
h. Next, the cells were incubated with the test compounds for 48 h. The cells were
then treated with cold PBS. After harvest, the cells were fixed in 70% ice-cold ethanol
overnight. Subsequently, the cells were centrifuged (1200 rpm for 5 min), the
supernatant was discarded and the pellet was treated with RNase A (100 ꢀg/ml) for 30
min at room temperature. The cells were stained using propidium iodide at a final
concentration of 50 ꢀg/mL. The stained cells were then analysed for cell cycle
distribution using flow cytometry (BECKMAN), and the changes in the cell cycle
profiles was analysed using CELL QUEST PRO.
5
.3 In vivo antitumor assay
Oxaliplatin was obtained from the Cancer Institute and Hospital, Chinese Academy
of Medical Sciences (CAMS). BALB/mice were obtained from the Institute for
Experimental Animals, Chinese Academy of Medical Sciences & Peking Union
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4

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Medical College, and animal care was in compliance with the regulations issued by
the Beijing Committee of Laboratory Animals.
5
.3.1 Exploration of maximum administration dosage
BALB/c mice were randomised into two groups (10 mice/group), and each group
was administered compound 1b dissolved in 10% DMSO via an intraperitoneal
injection at doses of 500 mg/kg and 1000 mg/kg, respectively. The mice were
monitored for visible signs of toxicity for 8 days.
5
.3.2 Antitumor efficiency assay
CT26 cancer cell suspensions were subcutaneously implanted into the right axilla
region of the mice. Next, the BALB/c mice were randomised into 5 groups (10
mice/group) that received the following treatments: blank control, 100 mg/kg or 50
mg/kg of 1b, 1.5 mg/kg of the positive control oxaliplatin or vehicle. The treatment
was initiated 24 h after tumor implantation, and the treatments were administered
once per day for ten days. Twenty-four hours after the final administration, the mice
were weighed and sacrificed, and the tumor s were excised, weighed and recorded for
analysis.
5
.4 Computational protocol
The pharmacophore was generated manually in Discovery studio 3.0 using the crystal
structure of Pip-1 complexed with MDM2 (PDB ID: 2 LZG) as reference and was
validated by calculating the ROC curve of the test set. Virtual screening was
performed in the “screen library” module, the “conformation generation” was set to
“fast”, and the other parameters were set as default. The obtained compounds were
further filtered by the “Rule of five”, and they were then subjected to docking analysis
using sybyl 7.3. The crystal structure with PDB ID 3LBL was prepared in sybyl 7.3
by removing the ligand and the water molecules and adding hydrogen atoms. The
docking parameters were all set as default. After the docking was completed, a visual
inspection for potential hit compounds was performed to identify the top 200
compounds ranked by docking score.
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The binding mode for compounds 4b, 1b and 2h were generated by molecular
docking using Autodock 4.2. The receptor and the ligands were prepared using
AutoDockTools and saved as pdbqt files. The centre of grid box was set to “-33.547,
1
6.037 -51.308”, and the number of points in the x, y and z dimensions were 46, 48
and 44, respectively. All the parameters for docking were set as default. After docking
was completed, PyMol was used to analyse the docking poses and generate
publication quality figures.
Acknowledgements
This work was supported by the Central Public-interest Scientific Institution Basal
Research Fund (2012IMBF05) and the National Mega-project for Innovative Drugs
(2012ZX09301002-001-017).
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